Direct identification of human oxytocin receptor-binding domains using a photoactivatable cyclic peptide antagonist: comparison with the human V1a vasopressin receptor.

Understanding of the molecular determinants responsible for antagonist binding to the oxytocin receptor should provide important insights that facilitate rational design of potential therapeutic agents for the treatment of preterm labor. To study ligand/receptor interactions, we used a novel photosensitive radioiodinated antagonist of the human oxytocin receptor, d(CH(2))(5) [Tyr(Me)(2),Thr(4),Orn(8),Phe(3(125)I,4N(3))-NH(2)9]vasotocin. This ligand had an equivalent high affinity for human oxytocin and V(1a) vasopressin receptors expressed in Chinese hamster ovary cells. Taking advantage of this dual specificity, we conducted photoaffinity labeling experiments on both receptors. Photolabeled oxytocin and V(1a) receptors appeared as a unique protein band at 70-75 kDa and two labeled protein bands at 85-90 and 46 kDa, respectively. To identify contact sites between the antagonist and the receptors, the labeled 70-75- and the 46-kDa proteins were cleaved with CNBr and digested with Lys-C and Arg-C endoproteinases. The fragmentation patterns allowed the identification of a covalently labeled region in the oxytocin receptor transmembrane domain III consisting of the residues Leu(114)-Val(115)-Lys(116). Analysis of contact sites in the V(1a) receptor led to the identification of the homologous region consisting of the residues Val(126)-Val(127)-Lys(128). Binding domains were confirmed by mutation of several CNBr cleavage sites in the oxytocin receptor and of one Lys-C cleavage site in the V(1a) receptor. The results are in agreement with previous experimental data and three-dimensional models of agonist and antagonist binding to members of the oxytocin/vasopressin receptor family.